Transcription Processivity: Protein-DNA Interactions Holding Together the Elongation Complex

The elongation of RNA chains during transcription occurs in a ternary complex containing RNA polymerase (RNAP), DNA template, and nascent RNA. It is shown here that elongating RNAP from Escherichia coli can switch DNA templates by means of end-to-end transposition without loss of the transcript. After the switch, transcription continues on the new template. With the use of defined short DNA fragments as switching templates, RNAP-DNA interactions were dissected into two spatially distinct components, each contributing to the stability of the elongating complex. The front (F) interaction occurs ahead of the growing end of RNA. This interaction is non-ionic and requires 7 to 9 base pairs of intact DNA duplex. The rear (R) interaction is ionic and requires approximately six nucleotides of the template DNA strand behind the active site and one nucleotide ahead of it. The nontemplate strand is not involved. With the use of protein-DNA crosslinking, the F interaction was mapped to the conserved zinc finger motif in the NH2-terminus of the β′ subunit and the R interaction, to the COOH-terminal catalytic domain of the β subunit. Mutational disruption of the zinc finger selectively destroyed the F interaction and produced a salt-sensitive ternary complex with diminished processivity. A model of the ternary complex is proposed here that suggests that trilateral contacts in the active center maintain the nonprocessive complex, whereas a front-end domain including the zinc finger ensures processivity.

[1]  Denise Grady Quick-Change Pathogens Gain an Evolutionary Edge , 1996, Science.

[2]  J. Berg,et al.  The Galvanization of Biology: A Growing Appreciation for the Roles of Zinc , 1996, Science.

[3]  S. Darst,et al.  Three-dimensional structure of E. coil core RNA polymerase: Promoter binding and elongation conformations of the enzyme , 1995, Cell.

[4]  R. Conaway,et al.  The RNA polymerase II elongation complex , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[5]  R. Weisberg,et al.  A zinc-binding region in the beta' subunit of RNA polymerase is involved in antitermination of early transcription of phage HK022. , 1995, Journal of molecular biology.

[6]  K. Severinov,et al.  Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  C. Chan,et al.  Discontinuous movements of DNA and RNA in RNA polymerase accompany formation of a paused transcription complex , 1995, Cell.

[8]  John Kuriyan,et al.  Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA , 1994, Cell.

[9]  K. Severinov,et al.  Topology of the product binding site in RNA polymerase revealed by transcript slippage at the phage lambda PL promoter. , 1994, The Journal of biological chemistry.

[10]  M. Kashlev,et al.  Discontinuous mechanism of transcription elongation. , 1994, Science.

[11]  R. Gourse,et al.  Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli. , 1993, The Journal of biological chemistry.

[12]  B. Alberts,et al.  The DNA replication fork can pass RNA polymerase without displacing the nascent transcript , 1993, Nature.

[13]  M. Kashlev,et al.  Active center rearrangement in RNA polymerase initiation complex. , 1993, The Journal of biological chemistry.

[14]  K. Severinov,et al.  Histidine-tagged RNA polymerase: dissection of the transcription cycle using immobilized enzyme. , 1993, Gene.

[15]  M. Chamberlin,et al.  Structural analysis of ternary complexes of Escherichia coli RNA polymerase. Deoxyribonuclease I footprinting of defined complexes. , 1992, Journal of molecular biology.

[16]  John Kuriyan,et al.  Three-dimensional structure of the β subunit of E. coli DNA polymerase III holoenzyme: A sliding DNA clamp , 1992, Cell.

[17]  P. V. von Hippel,et al.  The elongation-termination decision in transcription. , 1992, Science.

[18]  A. Sentenac,et al.  Zinc-binding subunits of yeast RNA polymerases. , 1991, The Journal of biological chemistry.

[19]  T. Kerppola,et al.  RNA polymerase: regulation of transcript elongation and termination , 1991, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[20]  R. Haselkorn,et al.  Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120 , 1991, Journal of bacteriology.

[21]  G. A. Rice,et al.  Footprinting analysis of mammalian RNA polymerase II along its transcript: an alternative view of transcription elongation. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[22]  P. V. von Hippel,et al.  A thermodynamic analysis of RNA transcript elongation and termination in Escherichia coli. , 1991, Biochemistry.

[23]  M. Chamberlin,et al.  RNA chain elongation by Escherichia coli RNA polymerase. Factors affecting the stability of elongating ternary complexes. , 1990, Journal of molecular biology.

[24]  R A Garrett,et al.  Archaebacterial DNA-dependent RNA polymerases testify to the evolution of the eukaryotic nuclear genome. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[25]  E. Zaychikov,et al.  Studies of the functional topography of Escherichia coli RNA polymerase. A method for localization of the sites of affinity labelling. , 1989, European journal of biochemistry.

[26]  R. Young,et al.  Prokaryotic and eukaryotic RNA polymerases have homologous core subunits. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[27]  D. Giedroc,et al.  Structural and functional differences between the two intrinsic zinc ions of Escherichia coli RNA polymerase. , 1986, Biochemistry.

[28]  R. Burgess,et al.  Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the λPR promoter , 1984 .

[29]  R. Burgess,et al.  A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. , 1975, Biochemistry.

[30]  M. Chamberlin,et al.  Ribonucleic acid chain elongation by Escherichia coli ribonucleic acid polymerase. I. Isolation of ternary complexes and the kinetics of elongation. , 1974, The Journal of biological chemistry.

[31]  A. Das Control of transcription termination by RNA-binding proteins. , 1993, Annual review of biochemistry.

[32]  Keith Smith Solid Supports and Catalysts in Organic Synthesis , 1992 .

[33]  M. Imperiale,et al.  RNA 3' end formation in the control of gene expression. , 1987, Annual review of genetics.

[34]  T Platt,et al.  Transcription termination and the regulation of gene expression. , 1986, Annual review of biochemistry.

[35]  K. E. J. Barrett,et al.  Dispersion polymerization in organic media , 1974 .